Immune Evasion and Recognition of the Syphilis Spirochete in Blood and Skin of Secondary Syphilis Patients: Two Immunologically Distinct Compartments

<div><h3>Background</h3><p>The clinical syndrome associated with secondary syphilis (SS) reflects the propensity of <em>Treponema pallidum</em> (<em>Tp</em>) to escape immune recognition while simultaneously inducing inflammation.</p> <h3>Methods</h3><p>To better understand the duality of immune evasion and immune recognition in human syphilis, herein we used a combination of flow cytometry, immunohistochemistry (IHC), and transcriptional profiling to study the immune response in the blood and skin of 27 HIV(-) SS patients in relation to spirochetal burdens. <em>Ex vivo</em> opsonophagocytosis assays using human syphilitic sera (HSS) were performed to model spirochete-monocyte/macrophage interactions <em>in vivo</em>.</p> <h3>Results</h3><p>Despite the presence of low-level spirochetemia, as well as immunophenotypic changes suggestive of monocyte activation, we did not detect systemic cytokine production. SS subjects had substantial decreases in circulating DCs and in IFNγ-producing and cytotoxic NK-cells, along with an emergent CD56−/CD16+ NK-cell subset in blood. Skin lesions, which had visible <em>Tp</em> by IHC and substantial amounts of <em>Tp</em>-DNA, had large numbers of macrophages (CD68+), a relative increase in CD8+ T-cells over CD4+ T-cells and were enriched for CD56+ NK-cells. Skin lesions contained transcripts for cytokines (IFN-γ, TNF-α), chemokines (CCL2, CXCL10), macrophage and DC activation markers (CD40, CD86), Fc-mediated phagocytosis receptors (FcγRI, FcγR3), IFN-β and effector molecules associated with CD8 and NK-cell cytotoxic responses. While HSS promoted uptake of <em>Tp</em> in conjunction with monocyte activation, most spirochetes were not internalized.</p> <h3>Conclusions</h3><p>Our findings support the importance of macrophage driven opsonophagocytosis and cell mediated immunity in treponemal clearance, while suggesting that the balance between phagocytic uptake and evasion is influenced by the relative burdens of bacteria in blood and skin and the presence of <em>Tp</em> subpopulations with differential capacities for binding opsonic antibodies. They also bring to light the extent of the systemic innate and adaptive immunologic abnormalities that define the secondary stage of the disease, which in the skin of patients trends towards a T-cell cytolytic response.</p> </div

Emergence of a CD56-negative NK-cell population in secondary syphilis (SS) patients.

<p>(<b>A</b>) The CD56^negative CD16+ NK-cells subset in untreated SS patients are shown by the black arrows. (<b>B</b>) Significant increases (* <i>p</i> = 0.003) in CD56^negative CD16+ NK-cell population was seen in SS patients. This anomaly was not present in healthy controls.</p

Transcriptional Profile - Secondary syphilis skin biopsies (n = 12) vs healthy control skin (n = 3).

<p>Transcriptional Profile - Secondary syphilis skin biopsies (n = 12) vs healthy control skin (n = 3).</p

Cell surface activation markers in secondary syphilis patient monocytes.

<p>Cell surface activation markers were examined in monocytes from secondary syphilis (SS) patients before (Pre-Tx) and around 60 days after penicillin treatment (Post-Tx) and compared to healthy controls. (<b>A</b>) A modest but significant decrease in CD40 MFI was evident between paired acute and convalescent samples obtained from SS patients before and after treatment. (<b>B</b>) Significant increases in CD14 MFI expression were observed between syphilis patients and healthy volunteers prior to antibiotic treatment and between pre- and post-penicillin treatment (<i>p</i> values are shown in the figure).</p

Immunophenotypic cellular composition of the inflammatory infiltrate in secondary syphilis patient skin lesions.

<p>IHC staining depicts CD68+ macrophages (<b>A</b> and <b>B</b>), CD4+ T-cells (<b>C</b> and <b>D</b>), CD8+ T-cells (<b>E</b> and <b>F</b>) and CD56+ NK-cells (<b>G</b> and <b>H</b>). Panels B, D, E and H are high magnification images of the red boxed areas in A, C, E and G.</p

Clinical and laboratory characteristics of secondary syphilis patients.

*<p>Diagnostic PCR results previously published in PLoS NTD (Ref 8).</p

Immunophenotypic alterations in dendritic cell (DC) populations.

<p>Circulating DCs were analyzed by flow cytometry in secondary syphilis (SS) patients before (Pre-Tx) and after penicillin treatment (Post-Tx). DCs were characterized by flow cytometry parameters as being HLA-DR+ and Lineage cocktail negative (not shown) and the expression of CD11c into monocytoid (CD11c+) and plasmacytoid (CD11c−) and expression of the co-stimulatory molecule CD83. A marked decrease was observed in the CD11c+ population in the blood of 7/12 SS patients and at the follow-up visit this population recovered in these same SS patients.</p

Secondary syphilis (SS) patients exhibit a significant decrease in total NK-cell populations.

<p>(<b>A</b>) Gating procedure to determine NK-cells subsets according to CD56 and CD16 expression by flow cytometry are shown. (<b>B and C</b>) A significantly larger percentage of SS subjects exhibit NK-cell values below the 5^th percentile of established published normal values (line depicts the cutoff), when compared to healthy controls (* indicates <i>p</i><0.05).</p